Proximity Labelling to Quantify Kv7.4 and Dynein Protein Interaction in Freshly Isolated Rat Vascular Smooth Muscle Cells

Understanding protein–protein interactions is crucial for unravelling subcellular protein distribution, contributing to our understanding of cellular organisation. Moreover, interaction studies can reveal insights into the mechanisms that cover protein trafficking within cells. Although various techniques such as Förster resonance energy transfer (FRET), co-immunoprecipitation, and fluorescence microscopy are commonly employed to detect protein interactions, their limitations have led to more advanced techniques such as the in situ proximity ligation assay (PLA) for spatial co-localisation analysis. The PLA technique, specifically employed in fixed cells and tissues, utilises species-specific secondary PLA probes linked to DNA oligonucleotides. When proteins are within 40 nm of each other, the DNA oligonucleotides on the probes interact, facilitating circular DNA formation through ligation. Rolling-circle amplification then produces DNA circles linked to the PLA probe. Fluorescently labelled oligonucleotides hybridise to the circles, generating detectable signals for precise co-localisation analysis. We employed PLA to examine the co-localisation of dynein with the Kv7.4 channel protein in isolated vascular smooth muscle cells from rat mesenteric arteries. This method enabled us to investigate whether Kv7.4 channels interact with dynein, thereby providing evidence of their retrograde transport by the microtubule network. Our findings illustrate that PLA is a valuable tool for studying potential novel protein interactions with dynein, and the quantifiable approach offers insights into whether these interactions are changed in disease.


Recipes
Prepare all solutions in volumetric flasks up to the specified volume.Using volumetric flasks will contribute to precise measurements and reliable results in your experiments.

Calcium chloride (1 M)
Reagent Final concentration Quantity or Volume Calcium chloride 1 M 11.098 g Dissolve the calcium chloride in Milli-Q water, add the volume up to exactly 100 mL, and store at 4 °C.

Calcium chloride (100 mM)
Dilute 100 μL of calcium chloride 1 M in 900 μL of Milli-Q water and store in a 1.5 mL tube at room temperature.

Magnesium chloride (1 M) Reagent
Final concentration Quantity or Volume Magnesium chloride hexahydrate 1 M 20.33 g Dissolve the magnesium chloride hexahydrate in Milli-Q water, add the volume up to exactly 100 mL, and store at 4 °C.

Reagent
Final 1 mM 200 μL Dissolve the reagents in Milli-Q water, adjust the pH to 7.4, and add the volume up to exactly 200 mL.The solution should be freshly prepared on the day of experiment.19 g Dissolve the reagents in Milli-Q water, adjust the pH to 7.4, and add up to exactly 500 mL.After preparation, distribute the solution into 50 mL tubes and store them at -20 °C.Thaw one 50 mL SMDS solution on the day of the experiment before use.

Wash buffer A and wash buffer B Published: Mar 20, 2024
Prepare PLA wash buffer A and B ahead by dissolving the PLA buffer sachets each in 1 L of Milli-Q.Filter sterilise both buffers using a syringe and a 0.2 μm pore filter and store at 4 °C.3. Cut the intestines just above the rectum and below the stomach and place the intestines in a beaker filled with HEPES-Krebs placed on ice (Figure 2A-2D).4. Place the intestines into a dissection dish filled with cold HEPES-Krebs buffer and pin the jejunum and ileum along the side of the dissection dish forming a circle, allowing visualisation of the mesenteric vessels, with the main arterial branch in the centre (Figure 2E).a. Arteries can be differentiated from veins by their thicker smooth muscle layer, which makes them more rigid compared to veins.Furthermore, the thicker wall creates the appearance of a reduced blood volume compared to veins.6. Remove all tissue surrounding the arteries (Figure 2G-2H).

Laboratory supplies
a. Begin by locating the larger veins and carefully tear them using two forceps; then, remove them all the way down to the lower-order branches.b.Cut through the mesentery connecting the arterial branches and remove all adipose tissue surrounding the arteries.7. Remove the mesenteric arteries by first cutting at the lower-order branches and then free the mesenteric arteries by cutting the central branch (Figure 2I-2K).8. Take a small section of the main artery, including 3-4 branches of lower-order mesenteric arteries and place it in a 1.5 mL tube filled with SMDS solution on ice.9. Remove the remaining intestines from the dissection dish for disposal.

C. Isolation of the mesenteric arterial smooth muscle cells
1. Have a fire-polished glass Pasteur pipette ready.To make a fire-polished pipette, break off the long end of the pipette and throw it out.Expose the pipette with its broken end to a Bunsen flame, constantly rotating it.Continue with this process until the edges are polished and the opening is smoothed and narrowed to an inner diameter of roughly 0.7-1 mm (Figure 1).Wash the pipette with ethanol after each experiment so it can be used for subsequent future experiments.2. The following solutions used for smooth muscle cell isolation have been adapted from Zhong et al. [14] and Chadha et al. [11].Prepare the following stock solutions (Recipes 7-9) by dissolving them in SMDS solution (Recipe 5) in 1.5 mL tubes and keep them at room temperature.a.These stock solutions have to be prepared fresh on the day of the experiment and cannot be stored for future experiments.3. Place the tube with the mesenteric artery branch in SMDS solution in a 37 °C heat block for 10 min to equilibrate the arteries at 37 °C.4. In the meantime, prepare Tube 1 (Recipe 8) by combining the stock solutions (Recipe 7) and SMDS solution in a 1.5 mL tube. 5. Transfer the mesenteric arteries to Tube 1. Gently invert the tube 4-5 times to ensure proper mixing of the arteries with the solution and incubate at 37 °C for 10 min.6.In the meantime, prepare Tube 2 (Recipe 9) by combining the stock solutions (Recipe 7) and SMDS solution in a 1.5 mL tube.7. Take the arteries from the heat block and wash five times in ice-cold SMDS solution using a glass Pasteur pipette with a rubber bulb.8. Transfer the mesenteric arteries to Tube 2. Gently invert the tube 4-5 times to mix the arteries with the solution, and incubate at 37 °C for 10 min.a.The arterial branch may appear slightly blurry or fluffy during this step; this means that the enzymatic digestion is effectively isolating the cells.9. Gently wash five times in ice-cold SMDS solution using a glass Pasteur pipette with a rubber cap.During the last wash, replace the solution with 500 μL of SMDS.10.Liberate the myocytes by gently triturating the arterial branch using the custom-made fire-polished glass pipette.a. Perform approximately 30 up-and-down triturations.11.Store the supernatant containing liberated myocytes in a separate tube on ice and add 500 μL of fresh SMDS to the arterial branch in the tube.Place a droplet of the supernatant on a glass slide and assess the cell density using a 10× microscope.12. Repeat the trituration process and save the supernatant in separate tubes on ice, until no more cells are released within the supernatant.a.This process can take approximately 1-5 rounds of trituration.Note: Consider shortening the artery incubation time (1-3 min shorter) with enzymes in tube 1 and tube 2, if the enzymes are newly opened, to prevent over-digestion of the arteries and enhance myocyte liberation.13.Combine the supernatants that have liberated myocytes and adjust the concentration of cells by adding more SMDS to achieve the desired cell density.By placing 50 µL of the cells on a glass coverslip, it is possible to check the cell density under a light microscope.There is no defined cell density necessary for undertaking this protocol, but smooth muscle cells should be visible under a light microscope's 20× objective, and the cells should not be crowded and touching one another.14.Add 1 μL of 100 mM calcium chloride for every 1 mL of cell suspension (resulting in a final concentration of 100 μM calcium chloride).Calcium is required for the cells to adhere to the coverslips.Note: Consider increasing the calcium chloride concentration by 10%-20% if there is significant loss of cells from the coverslip after fixing the cells.15.Add 100 μL of cell suspension on a 12 mm coverslip in a 24-well plate and keep at room temperature for 30 min to let the cells adhere to the coverslips (Figure 3A).a.After 30 min, the cells can be incubated with a pharmacological agent.To do this, dissolve the compound at the desired concentration in SMDS solution supplemented with 100 μM calcium chloride, replace the solution on the coverslip with 100 μL of compound solution, and incubate at 37 °C.16.Fix the cells by gently aspirating the SMDS solution from the coverslip and apply 400 μL of 4% PFA to each well.Place the 24-wells on the rocker for 15 min at room temperature.17.Wash three times with PBS, 400 μL per well.18. Use the coverslips immediately for PLA experiment or store coverslips in the 24-wells plate (with each well containing 1 mL of PBS and sealed with parafilm) at 4 °C for up to four weeks until used for the PLA experiment.The duration of storage depends on the specific cell type.

Part II: PLA
A. Preparation

B. Permeabilization, blocking, and primary antibody incubation
1. Prepare a sufficient volume of 0.1% Triton-X in PBS to add 400 μL of the solution to each coverslip within the well.2. Add 400 μL of 0.1% Triton-X to each well, place on the rocker, and incubate for 5 min to permeabilise the cells.3. Wash two times for 5 min each in PBS. 4. Remove the PBS and apply three drops of the blocking buffer (provided in the PLA probe kit) onto the cells on the coverslip in each well.Incubate at 37 °C for 30 min. 5. Prepare the primary antibody mix.It is very important that the two primary antibodies targeting the protein of interest are raised in different species.a.In our experiment, to look at Kv7.4 and dynein co-localisation, we prepared a primary antibody mixture consisting of anti-dynein and anti-Kv7.4antibodies.6. Dilute the primary antibodies in the antibody diluent provided in the PLA probe kit, following the specific requirements of each antibody.a. Prepare enough to use 40 μL of the primary antibody mix for each coverslip.7. Create a humidified chamber (Figure 3B).a. Use the lid of a Styrofoam ice box; the lid must have a recessed area in the middle.b.Place a piece of parafilm in the centre of the lid, where the coverslips will be placed for incubation.c.Create four wells using parafilm, each well containing a wet tissue inside (see Figure 3B).Place the four wet tissues in parafilm wells around the flat piece of parafilm in the middle of the Styrofoam lid.This will help maintain moisture and prevent the coverslips from drying out during the overnight incubation with the primary antibodies.8. Add a 40 μL drop of primary antibody mix to the parafilm.9. Using forceps, lift the coverslip from the blocking solution, removing it from the well.Gently tap the side of the coverslip on a paper tissue to remove excess blocking buffer and place it over the antibody drop with the side containing cells making contact with the drop and facing downward (Figure 3C).10.Cover the Styrofoam lid with cling film and leave it overnight at 4 °C (Figure 3D).
Note: This primary antibody concentration and incubation time may require optimisation.

C. Probe incubation
Note: Each coverslip represents one reaction, and 40 µL of solution is used per reaction.The calculations provided in the following steps are for a single reaction.
1.The following day, take the PLA PLUS and MINUS probe out of the fridge and vortex.2. Prepare the PLA probe mix by diluting the PLA PLUS and MINUS probes 1:5 in the antibody diluent provided in the kit.Make sufficient PLA probe solution to use 40 μL per coverslip.a.For one coverslip (40 μL): dilute 8 μL of PLUS and 8 μL of MINUS in 24 μL of antibody diluent.3. Place the coverslips incubated with the primary antibodies back in the 24-well plate and wash two times for 5 min each in PLA wash buffer A at room temperature on a rocker.4. Place a new piece of parafilm in the handcrafted humidified chamber and add 40 μL drops of PLA probe mix for every coverslip.5. Add the coverslips on top of the droplets with the side containing cells facing downward.6. Place the humidified chamber in a 37 °C incubator for 1 h.

D. Ligation
1. Place the coverslips back in the 24-well plate and wash two times for 5 min each in PLA wash buffer A at room temperature on a rocker.2. Prepare the PLA ligation mix by diluting the 5× ligation buffer with Milli-Q water (1:5 dilution) and add the ligase (1:40 dilution) to the mix.a. Include a negative control sample as part of the experiment.A suitable negative control may involve the use of two protein-targeting antibodies that are known to have no co-localisation and, therefore, should not produce a signal.Alternatively, consider using protein knockdown cells for additional control measures.Another appropriate control, as implemented in our study, involves the use of only one of the primary antibodies.

B. Data analysis
Note: Analyse the number of fluorescent PLA dots within each cell.Depending on the desired approach, PLA dots can be quantified on a single mid-cell z-plane or conducted on a maximum intensity projection encompassing the entire cell.
1. Open the image file in ImageJ.Make sure to open the images for PLA dots, DAPI, and brightfield in separate windows instead of using the merged channels file.You can achieve this by selecting the Split channel option (Figure 5A).
Published: Mar 20, 2024 number of PLA dots for one specific stack should be counted, select No, and ImageJ will count the dots for the stack you have currently selected in your PLA image window.9. To compare the average number of PLA dots for each cell under various conditions (such as control or pharmacologically treated cells), add the dot counts for each biological replicate into GraphPad PRISM.Analyse the data set using a nested t-test to test whether pharmacological treatment has an impact on the number of interactions between two proteins.

Published: Mar 20, 2024 Figure 1 .
Figure 1.Fire-polishing of the glass Pasteur pipette. A. Remove and discard the long end of the pipette.B. Rotate the end of the pipette in a Bunsen flame until its edges are polished and the opening is smoothed and narrowed.Comparison of the opening before (left) and after (right) fire polishing.

Software 1 . 7 Published 1 .
ImageJ (version 1.53k, July 2021) 2. GraphPad PRISM (version 9, October 2020) Procedure Part I: Isolation of rat vascular smooth muscle cells A. Preparation 1.Take a 50 mL Falcon tube with SMDS solution out of the -20 °C freezer and thaw on ice.2. Prepare the HEPES-Krebs solution (see Recipes) 3. Take the enzymes out of the -20 °C freezer and thaw at room temperature.Sacrifice the rat (in accordance with annex IV of the EU Directive 2010/63EU on the protection of animals used for scientific purposes).a. Make the rat unconscious by a single percussive blow to the head.Immediately after the onset of unconsciousness, perform cervical dislocation to complete the killing.2. Perform a laparotomy using surgical scissors and elevate the intestines out of the abdominal cavity (Figure 2A-2D).

Figure 2 .
Figure 2. Dissection process of rat mesenteric arteries.A. The male Wistar rat is euthanised via cervical dislocation.B-D.A laparotomy is performed, and the rat intestines are isolated.E. The intestines are pinned onto a dissection dish using sewing pins to expose the mesenteric vessels.F-H.The main mesenteric artery and vein, surrounded by adipose tissue, are visualised.I-K.The surrounding tissue is removed, and 3-4 branches of the mesenteric arteries are isolated for the extraction of arterial smooth muscle cells.

Figure 3 .
Figure 3. Adhesion and primary antibody incubation for isolated vascular smooth muscle cells.A. Place 100 µL of cell suspension onto each coverslip in the well.B. Create a humidified chamber using a Styrofoam icebox and parafilm.C. Incubate the cells with the primary antibody by applying a drop of the diluted antibody on the parafilm and placing the coverslip with adhered cells on top.D. Seal the humidified chamber with cling film for incubation at 4 °C.

Figure 4 .
Figure 4. Representative images of a single vascular smooth muscle cell displaying a number of proximity ligation assay (PLA) dots.Each red dot represents a site where the two primary antibodies have bound to their respective protein within 40 nm of each other.A. Texas Red channel (excitation wavelength 592), in which the PLA dots are visible.B. Brightfield image of the vascular smooth muscle cell.C. DAPI staining of the nucleus (excitation wavelength 353).D. Merged image of A, B, and C. Scale = 5 µm 2. Use appropriate microscope settings.a.In our specific setup, we capture images using a 63× oil immersion objective on either a ZEIS LSM780 or LSM900 laser scanning confocal microscope.b.Choose a DAPI filter for nucleus detection and a Texas red filter for PLA dot detection.To visualise cell contours and contrast, include a brightfield imaging channel in your microscopy setup (Figure4).c.Ensure consistent laser settings are applied for each experiment.3. Capture full z-stack images of individual cells.Multiple cells can be captured per coverslip.4. For quantitative comparisons, PLA signals of at least 30 cells are captured from at least three biological replicates.a. Include a negative control sample as part of the experiment.A suitable negative control may involve the use of two protein-targeting antibodies that are known to have no co-localisation and, therefore, should not produce a signal.Alternatively, consider using protein knockdown cells for additional control measures.Another appropriate control, as implemented in our study, involves the use of only one of the primary antibodies.

concentration Quantity or Volume
This protocol or parts of it has been used and validated in the following research article(s):  van der Horst et al. (2021).Dynein regulates Kv7.4 channel trafficking from the cell membrane.J. Gen. Physiol.(Figures 3F, 6C, 7B&C and 8A).